1. Field of the Invention
The present invention relates to an illumination switching apparatus and method for switching illumination for a sample through an objective between total reflection illumination and standard fluorescent observation illumination.
2. Description of the Related Art
Attention is now being paid to total internal reflection fluorescence microscopes (TIRFM). As shown in FIG. 12, total internal reflection fluorescence microscopes totally reflect light at the border between a cover slip 100 and sample 102. At this time, evanescent light, which occurs in a small region of several hundreds of nanometers or less in the sample 102, excites a fluorescent material in the sample. As a result, fluorescent light is generated in the small region near the cover slip 100. Through total internal reflection fluorescence microscopes, only this fluorescent light is observed. Since total internal reflection fluorescence microscopes provide a very dark background, observation with high contrast and observation using vary weak fluorescent light can be realized.
Evanescent light 103 does not reach the deep portion of the sample 102 located away from the cover slip 100. Accordingly, the deep portion cannot be observed.
Therefore, switching between observation using total reflection of fluorescent light and observation using standard fluorescent light enables the portion of the sample 102 near the cover slip 100 to be observed with high contrast, and also the entire sample 102 to be observed. In particular, when physiological phenomena of high reaction speed are being observed, prompt switching is required between observation using total reflection of fluorescent light and observation using standard fluorescent light.
Jpn. Pat. Appln. KOKAI Publications Nos. 9-159922 and 2002-31762, for example, disclose switching through an objective between observation using total reflection of fluorescent light and observation using standard fluorescent light. Specifically, Jpn. Pat. Appln. KOKAI Publication No. 9-159922 discloses a technique for parallel-moving an illumination light reflection mirror toward the objective, thereby moving the incident position of the light on the objective away from the optical axis of the lens, resulting in a change in the angle of emergence of light from the lens. Jpn. Pat. Appln. KOKAI Publication No. 2002-31762 discloses a technique for varying the angle of a mirror provided in the middle of the illumination system, thereby varying the angle of emergence of light from the objective.
However, in the structure of Jpn. Pat. Appln. KOKAI Publication No. 9-159922, in which the mirror provided in the middle of the illumination system is moved parallel, or in the structure of Jpn. Pat. Appln. KOKAI Publication No. 2002-31762, in which the angle of the mirror is varied, the single mirror must be reciprocated between precisely determined positions when switching is performed between observation using total reflection of fluorescent light and observation using standard fluorescent light.
Such mirror reciprocation makes it difficult to enhance the accuracy of the position at which the mirror is stopped, in proportion to the speed of switching. Unless the mirror stop position accuracy can be enhanced, when switching is performed twice, the observation image before switching cannot be exactly reproduced.
If illumination light has a large cross section at the mirror position, a large mirror is used. This imparts a limit on the speed of switching between observation using total reflection of fluorescent light and observation using standard fluorescent light. The switching period employed where the mirror is moved parallel is, for example, 0.5 sec. at minimum. On the other hand, the switching period employed where the mirror angle is varied is, for example, 0.1 sec. at minimum. Thus, both cases do not satisfy the demand for a short switching period of the order of 1/100 sec. needed for switching between observation using total reflection of fluorescent light and observation using standard fluorescent light.